Basic Informations

C.V

Master Title

EFFECTS OF CERTAIN AGENTS ON IATROGENIC HEPATOTOXICITY IN EXPERIMENTAL ANIMALS

Master Abstract

In the present study, the hepatotoxic effects of four drugs in current medical use, namely paracetamol, phenobarbitone, isoniazid, and rifampicin, were investigated in experimental rats. Three successful submaximal hepatotoxicity models were constructed, based on the doses reported in the literature as well as on pilot experimental trials. These are paracetamol, paracetamol-phenobarbitone, and isoniazid-rifampicin hepatotoxicity models. For setting of the first model, paracetamol was administered in single intraperitoneal doses of 500-700 mg/kg to adult fasted and fed rats, and in single oral doses of 400-1000 mg/kg to adult fasted rats. Oral doses of 600 and 800 mg/kg, administered to fasted adult rats, were selected as submaximal hepatotoxic paracetamol doses. In the second model, phenobarbitone was co-administered with paracetamol to adult rats. Phenobarbitone was administered via the intraperitoneal route, in a dose of 75 mg/kg/day for three consecutive days, followed by a single oral dose of paracetamol, 600 mg/kg, on fourth day. For setting of the third model, isoniazid and rifampicin were individually administered to young rats via the intraperitoneal route, each in a dose of 50 mg/kg/day for 21 days, and co-administered in intraperitoneal doses of 50 and 100 mg/kg/day of each drug for 21 days. Isoniazid-rifampicin co-administration to young rats (each in a dose of 50 mg/kg/day, i.p., 21 days) was selected to represent submaximal hepatotoxicity. Adult rats were fasted 18 hours before paracetamol administration and remained fasted for 24 hours after paracetamol administration then sacrificed. Young rats were deprived of food 12 hours after the last drug administration, remained fasted for 12 hours, then sacrificed. In all models, animals were sacrificed 24 hours after the last hepatotoxic dose administration. Three drugs, thought to act through different protective mechanisms, were studied to assess their hepatoprotective potentials in the previously-mentioned hepatotoxicity models. These are N-acetylcysteine as an antioxidant and glutathione precursor, cimetidine as a liver microsomal enzyme inhibitor, and nifedipine as a calcium channel blocker. To study the effect of N-acetylcysteine in normal adult rats, it was administered in a single oral dose of 1200 mg/kg. When it was studied for possible hepatoprotective effect against paracetamol-induced hepatotoxicity, it was administered in single oral doses of 500 and 1200 mg/kg, one hour before paracetamol. To explore the effect of N-acetylcysteine on normal young rats, it was administered in a dose of 100 mg/kg/day, i.p., for 21 days. To study the possible hepatoprotective effect against isoniazid-rifampicin-induced hepatotoxicity, it was given in repetitive intraperitoneal doses of 50 and 100 mg/kg/day for 21 days, parallel to isoniazid and rifampicin. To investigate the effect of cimetidine on normal adult rats, it was administered in two consecutive doses, each of 100 mg/kg, i.p., with 2-hour interval. When it was tested as a hepatoprotectant against paracetamol- and paracetamol-phenobarbitone-induced hepatotoxicities, it was administered in a single intraperitoneal dose of 100 mg/kg one hour before paracetamol, or in two consecutive intraperitoneal doses, each of 100 mg/kg, given one hour before and one hour after paracetamol. To study the effect of cimetidine on normal young rats, it was administered repetitively in a dose of 40 mg/kg/day, i.p., for 21 days. When tested against isoniazid-rifampicin-induced hepatotoxicity, it was administered in repetitive intraperitoneal doses of 20 and 40 mg/kg/day, 21 days, parallel to isoniazid and rifampicin. When the effect of nifedipine was explored in normal adult rats, it was administered in two consecutive doses, each of 25 mg/kg, i.p., with 8-hour interval. When tested against paracetamol-induced hepatotoxicity, nifedipine was administered in a single intraperitoneal dose of 25 mg/kg one hour before paracetamol, or in two consecutive intraperitoneal doses, each of 25 mg/kg, given one hour before and seven hours after paracetamol. To investigate the effect of nifedipine in normal young rats, it was administered repetitively in a dose of 10 mg/kg/day, i.p., for 21 days. When tested against isoniazid-rifampicin-induced hepatotoxicity, it was given in repetitive intraperitoneal doses of 5 and 10 mg/kg/day, 21 days, parallel to isoniazid and rifampicin. Serum activities of glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), and lactate dehydrogenase (LDH) enzymes were estimated as biomarkers of liver injury. Serum malondialdehyde (MDA) level, hepatic glutathione (GSH) content, and hepatic cytosolic glutathione-S-transferase (GST) activity were measured as oxidative stress biomarkers. Liver calcium content and liver weight/body weight ratio were estimated as parameters related to hepatotoxic calcium deregulation and inflammation, respectively. The biochemical estimations were confirmed by a histopathological study. The main findings of the present investigation can be summarized as follows: 1. Acute oral N-acetylcysteine administration to normal adult rats (1200 mg/kg, p.o., single dose), or repetitive intraperitoneal administration to normal young rats (100 mg/kg/day, i.p., 21 days), did not significantly affect serum GPT activity, liver GSH and calcium contents, liver cytosolic GST activity and liver weight/body weight ratio. 2. Acute intraperitoneal cimetidine administration to normal adult rats (100 mg/kg, i.p., twice with 2-hour interval), or repetitive administration to young rats (40 mg/kg/day, i.p., 21 days), resulted in significant elevations of serum GPT activity, but did not significantly affect liver GSH and calcium contents, liver cytosolic GST activity, and liver weight/body weight ratio. 3. Acute intraperitoneal nifedipine administration to adult rats (25 mg/kg, i.p., twice with 8-hour interval) resulted in a significant elevation of serum GPT activity, but did not significantly affect liver GSH and calcium contents, liver cytosolic GST activity, and liver weight/body weight ratio. Repetitive intraperitoneal nifedipine administration to young rats (10 mg/kg/day, i.p., 21 days) did not significantly affect serum GPT activity, liver GSH and calcium contents, liver cytosolic GST activity, and liver weight/body weight ratio. 4. Acute administration of paracetamol to adult fasted rats in a single intraperitoneal dose of 500 mg/kg resulted in significant elevations of serum GPT and GOT activities but did not significantly affect liver weight/body weight ratio. Acute administration of paracetamol to adult fasted rats in a single intraperitoneal dose of 700 mg/kg resulted in significant increases of serum GPT and GOT activities as well as liver weight/body weight ratio. Acute administration of paracetamol in single oral doses of 500 and 700 mg/kg to fed rats did not significantly affect serum GPT and GOT activities as well as liver weight/body weight ratio. 5. Oral and intraperitoneal routes of paracetamol administration to adult fasted rats in a single dose of 500 mg/kg did not significantly differ regarding serum GPT and GOT activities as well as liver weight/body weight ratio. Oral and intraperitoneal routes of paracetamol administration in a single dose of 700 mg/kg showed significant differences with respect to serum GPT and GOT activities but not liver weight/body weight ratio. 6. Acute administration of paracetamol to adult fasted rats in a single oral dose of 400 mg/kg did not significantly affect serum GPT and GOT activities, serum MDA level, hepatic GSH content, and liver weight/body weight ratio. 7. Acute administration of paracetamol to adult fasted rats in single oral doses of 500-1000 mg/kg significantly increased serum GPT, GOT and LDH activities, significantly increased serum MDA level and hepatic calcium content, significantly decreased hepatic GSH content and hepatic cytosolic GST activity, and resulted in the occurrence of histopathological lesions, but did not significantly affect liver weight/ body weight ratio in doses up to 800 mg/kg. 8. N-acetylcysteine administration (500, 1200 mg/kg, p.o.) one hour before paracetamol (600 mg/kg, p.o.) significantly lowered paracetamol-induced rises of serum GPT, GOT, and LDH activities, significantly lowered paracetamol-induced elevations of serum MDA level and hepatic calcium content, and significantly improved paracetamol-induced suppression of hepatic GSH content and hepatic cytosolic GST activity, but did not significantly affect liver weight/body weight ratio of paracetamol-treated rats. N-acetylcysteine administration (500, 1200 mg/kg, p.o.) improved liver histopathology of paracetamol-treated rats. 9. N-acetylcysteine administration (500 mg/kg, p.o.) one hour before paracetamol (800 mg/kg, p.o.) significantly lowered paracetamol-induced rises of serum GPT and GOT activities as well as paracetamol-induced elevation of serum MDA level, but did not significantly affect hepatic GSH content and liver weight/body weight ratio of paracetamol-treated rats. N-acetylcysteine administration (1200 mg/kg, p.o.) one hour before paracetamol (800 mg/kg, p.o.) significantly lowered paracetamol-induced rises of serum GPT and GOT activities and paracetamol-induced elevation of serum MDA level, and significantly improved paracetamol-induced suppression of hepatic GSH content, but did not significantly affect liver weight/body weight ratio of paracetamol-treated rats. 10. Cimetidine administration (100 mg/kg, i.p.) one hour before, or one hour before and one hour after, paracetamol (600 mg/kg, p.o.) did not significantly affect serum GPT, GOT, and LDH activities, serum MDA level, liver GSH and calcium contents, liver cytosolic GST activity, and liver weight/body weight ratio of paracetamol-treated rats. 11. Nifedipine administration (25 mg/kg, i.p.) one hour before paracetamol (600 mg/kg) did not significantly affect serum GPT and GOT activities, liver GSH content, and liver weight/body weight ratio of paracetamol-treated rats. Nifedipine administration (25 mg/kg, i.p.) one hour before and seven hours after paracetamol (600 mg/kg) significantly decreased paracetamol-induced rises of serum GPT, GOT, and LDH activities, as well as paracetamol-induced rises of serum MDA level and hepatic calcium content. Nifedipine administration (25 mg/kg, i.p.) one hour before and seven hours after paracetamol (600 mg/kg) did not significantly affect hepatic GSH content, hepatic cytosolic GST activity, and liver weight/body weight ratio of paracetamol-treated rats, but improved liver histopathology. 12. Nifedipine administration (25 mg/kg, i.p.) one hour before and seven hours after paracetamol (800 mg/kg) did not significantly affect serum GPT and GOT activities, serum MDA level, liver GSH content, and liver weight/body weight ratio of paracetamol-treated rats. 13. Repetitive short-term intraperitoneal administration of phenobarbitone to adult rats (75 mg/kg/day, 3 days) significantly increased liver cytosolic GST activity, slightly affected liver histopathology, and did not significantly affect serum GPT, GOT, and LDH activities, serum MDA level, hepatic GSH and calcium contents, and liver weight/body weight ratio. 14. Paracetamol (600 mg/kg, p.o.)-phenobarbitone (75 mg/kg, i.p., 3 days)-co-administration significantly increased serum GPT, GOT, and LDH activities, significantly increased serum MDA level, liver calcium content, and liver weight/body weight ratio, significantly decreased liver GSH content, and dramatically affected liver histopathology, but did not significantly affect liver cytosolic GST activity. 15. Phenobarbitone administration (75 mg/kg/day, i.p., 3 days) significantly potentiated paracetamol (600 mg/kg, p.o.)-induced rises of serum GPT and GOT activities, as well as paracetamol-induced elevation of serum MDA level. 16. Cimetidine administration (100 mg/kg, i.p.) to paracetamol (600 mg/kg, p.o.)-phenobarbitone (75 mg/kg/day, i.p., 3 days) co-treated rats, one hour before paracetamol, significantly decreased paracetamol-phenobarbitone-induced elevation of serum GOT activity, but did not significantly affect serum GPT activity of paracetamol-phenpbarbitone co-treated rats. 17. Cimetidine administration (100 mg/kg, i.p.) to paracetamol (600 mg/kg, p.o.)-phenobarbitone (75 mg/kg/day, i.p., 3 days) co-treated rats, in two consecutive doses given one hour before and one hour after paracetamol, significantly decreased paracetamol-phenobarbitone-induced rises of serum GPT, GOT, and LDH activities, significantly reduced paracetamol-phenobarbitone-induced elevations of serum MDA level and hepatic calcium content, and significantly increased paracetamol-phenobarbitone-induced suppressions of hepatic GSH content and hepatic cytosolic GST activity. Cimetidine administration (100 mg/kg, i.p.) to paracetamol (600 mg/kg, p.o.)-phenobarbitone (75 mg/kg/day, i.p., 3 days) co-treated rats, in two consecutive doses given one hour before and one hour after paracetamol, improved liver histopathology but did not significantly affect liver weight/body weight ratio of paracetamol-phenobarbitone co-treated rats. 18. Repetitive long-term administration of isoniazid alone (50 mg/kg/day, i.p.) or rifampicin alone (50 mg/kg/day, i.p.) to young rats for 21 days did not significantly affect serum GPT and GOT activities, liver GSH content, and liver weight/body weight ratio. 19. Repetitive long-term co-administration of isoniazid (50 mg/kg/day, i.p.) and rifampicin (50 mg/kg/day, i.p.) to young rats for 21 days significantly increased serum GPT, GOT and LDH activities, significantly elevated serum MDA level, significantly lowered hepatic GSH content and hepatic cytosolic GST activity, and resulted in the occurrence of histopathological lesions, but did not significantly affect liver weight/body weight ratio or liver calcium content. Repetitive long-term co-administration of isoniazid (100 mg/kg/day, i.p.) and rifampicin (100 mg/kg/day, i.p.) to young rats for 21 days significantly raised serum GPT, GOT and LDH activities, significantly increased serum MDA level, significantly suppressed hepatic GSH content and hepatic cytosolic GST activity, significantly increased liver weight/body weight ratio, and resulted in the occurrence of histopathological lesions, but did not significantly affect liver calcium content. 20. N-acetylcysteine administration (50 mg/kg/day, i.p., 21 days) to isoniazid (50 mg/kg/day, i.p., 21 days)-rifampicin (50 mg/kg/day, i.p., 21 days) co-treated rats did not significantly affect serum GPT and GOT activities, liver GSH content, and liver weight/body weight ratio, and did not improve liver histopathology. 21. N-acetylcysteine administration (100 mg/kg/day, i.p., 21 days) to isoniazid (50 mg/kg/day, i.p., 21 days)-rifampicin (50 mg/kg/day, i.p., 21 days) co-treated rats significantly decreased isoniazid-rifampicin-induced elevations of serum GPT, GOT, and LDH activities, significantly lowered isoniazid-rifampicin-induced elevations of serum MDA level, significantly improved isoniazid-rifampicin-induced suppression of hepatic GSH content and hepatic cytosolic GST activity, and improved liver histopathology, but did not significantly affect liver weight/body weight ratio and liver calcium content. 22. Cimetidine administration (20 mg/kg/day, i.p., 21 days) to isoniazid (50 mg/kg/day, i.p., 21 days)-rifampicin (50 mg/kg/day, i.p., 21 days) co-treated rats significantly decreased isoniazid-rifampicin-induced elevations of serum GPT and GOT activities and improved liver histopathology, but did not significantly affect liver GSH content and liver weight/body weight ratio. 23. Cimetidine administration (40 mg/kg/day, i.p., 21 days) to isoniazid (50 mg/kg/day, i.p., 21 days)-rifampicin (50 mg/kg/day, i.p., 21 days) co-treated rats significantly decreased isoniazid-rifampicin-induced elevations of serum GPT, GOT, and LDH activities, significantly lowered isoniazid-rifampicin-induced elevations of serum MDA level, significantly improved isoniazid-rifampicin-induced suppression of liver GSH content, and improved liver histopathology, but did not significantly affect liver calcium content, liver cytosolic GST activity, and liver weight/body weight ratio. 24. Nifedipine administration (5 mg/kg/day, i.p., 21 days) to isoniazid (50 mg/kg/day, i.p., 21 days)-rifampicin (50 mg/kg/day, i.p., 21 days) co-treated rats did not significantly affect serum GPT and GOT activities, liver GSH content, and liver weight/body weight ratio. 25. Nifedipine administration (10 mg/kg/day, i.p., 21 days) to isoniazid (50 mg/kg/day, i.p., 21 days)-rifampicin (50 mg/kg/day, i.p., 21 days) co-treated rats significantly increased serum GPT, GOT, and LDH activities and significantly suppressed liver GSH content, but did not significantly affect serum MDA level, liver calcium content, liver cytosolic GST activity, and liver weight/body weight ratio. CONCLUSION: According to the results of the present study, we can conclude the following: 1. Paracetamol and isoniazid are true hepatotoxic agents. A common feature of the two drugs is that they are both converted to reactive toxic electrophilic metabolites by the liver microsomal CYP450 enzyme system, and the metabolites bind to hepatic GSH and then to hepatocellular macromolecules, yielding their hepatotoxic effects. Another common feature is that the hepatotoxicities of both drugs are associated with oxidative stress and compromised cellular antioxidant defense mechanisms. The reactive metabolites initiate lipid peroxidation either directly or through reactive oxygen species. They differ in the sense that paracetamol hepatotoxicity, unlike isoniazid hepatotoxicity, is associated with dramatic increases of liver calcium content. 2. Phenobarbitone and rifampicin are not considered true hepatotoxins, and their hepatotoxicities are mainly attributed to their ability to stimulate liver microsomal enzymes and hence to potentiate the hepatotoxicities of the true hepatotoxins, paracetamol and isoniazid, respectively, via stimulating the production of their reactive toxic metabolites. 3. Paracetamol is hepatotoxic to adult fasted rats in a single oral or intraperitoneal dose administration. Fasting is essential for the induction of paracetamol hepatotoxicity in experimental rats. Oral and intraperitoneal routes of administration show comparable hepatotoxicities. A threshold hepatotoxic paracetamol dose exists, below which paracetamol shows no appreciable hepatotoxicity. 4. Paracetamol-phenobarbitone and isoniazid-rifampicin combinations are hepatotoxic to adult and young rats in acute and long-term dose regimens, respectively. 5. N-acetylcysteine exhibits a significant hepatoprotective effect against paracetamol-induced hepatotoxicity in adult rats as well as against isoniazid-rifampicin-induced hepatotoxicity in young rats. The hepatoprotective effect is most probably attributed to its antioxidant effect, either directly or through stimulation of hepatic GSH biosynthesis. That is, antioxidant therapy seems to be an effective tool against paracetamol- and isoniazid-induced hepatotoxicities. 6. Cimetidine is hepatoprotective against paracetamol-phenobarbitone-induced hepatotoxicity in adult rats, but not against paracetamol-induced hepatotoxicity in adult rats. It is also hepatoprotective against isoniazid-rifampicin-induced hepatotoxicity in young rats. In either case, the hepatoprotective effect is most probably attributed to the potent liver microsomal enzyme inhibitory effect of cimetidine. The effect is evident only when liver microsomal enzymes that are in the inhibitory spectrum of cimetidine, and that are responsible for the biotransformation of the hepatotoxins to their toxic metabolites, are involved. This is the case in paracetamol-phenobarbitone-induced and isoniazid-rifampicin-induced hepatotoxicities, where CYP3A4 is most probably the involved subfamily. That is, liver microsomal enzyme inhibition may be an effective hepatoprotective tool against drugs whose hepatotoxicities depend on their biotransformation to reactive toxic metabolites. 7. Nifedipine exhibits a significant hepatoprotective effect against paracetamol-induced hepatotoxicity in adult rats. On the other hand, nifedipine is not hepatoprotective against isoniazid-rifampicin-induced hepatotoxicity in young rats. The hepatoprotective effect against paracetamol-induced hepatotoxicity is most probably attributed to the control of calcium homeostasis and inhibition of cellular calcium influx. That is, calcium channel blockers may have significant hepatoprotective activities only against drugs whose hepatotoxicities are associated with calcium deregulation. 8. Nifedipine administration significantly potentiates isoniazid-rifampicin-induced hepatotoxicity in young rats. The effect is most probably attributed to the liver microsomal CYP3A4 enzyme induction together with the lack of a hepatoprotective effect. In case of paracetamol-induced hepatotoxicity, however, the hepatoprotective effect of nifedipine, through inhibition of calcium influx, may overwhelm the hepatotoxic effect of the drug through CYP450 induction. 9. Compared to cimetidine and nifedipine, N-acetylcysteine is a safer hepatoprotective and has a wider hepatoprotective spectrum.

PHD Title

PHARMACOLOGICAL STUDY OF THE POSSIBLE PROTECTIVE EFFECTS OF SOME ANTIOXIDANTS AGAINST EXPERIMENTALLY-INDUCED HEPATOTOXICITY IN RATS

PHD Abstract

In the present investigation, the possible hepatoprotective effects of three natural extracts, namely aloe vera leaf pulp extract, grape seed extract and nutmeg extract were studied in comparison with N-acetylcysteine (NAC) as a standard hepatoprotective agent. To achieve this goal, two models of hepatotoxicity were performed; an in vivo model of rat hepatic ischemia/reperfusion (IR) injury and an in vitro model using freshly-isolated rat hepatocyte suspension incubated with carbon tetrachloride (CCl4) as a chemical hepatotoxicant. For setting of the in vivo model, hepatic IR was performed by clamping the portal triad (hepatic artery, portal vein and bile duct) of fasted adult male albino rats (one group was left as sham-operated control operation) for different time periods (15 or 30 minutes) followed by reperfusion (15, 30 or 60 minutes). According to the obtained results, hepatic IR injury model was set as 30 minutes of ischemia followed by 30 minutes of reperfusion. Test agents (NAC: 150, 300 and 600 mg/kg/day; aloe extract: 5, 10 and 20 ml/kg/day; grape seed extract: 200, 400 and 800 mg/kg/day and nutmeg extract: 250, 500 and 1000 mg/kg/day) were administered orally on daily basis for seven days to normal animals to check the presence of any effect on normal liver functions and to choose suitable doses to conduct in the current experiments. According to the published literature as well as pilot trials, all test agents (NAC 300 mg/kg/day, aloe 10 ml/kg/day, grape seed 400 mg/kg/day and nutmeg 500 mg/kg/day) were administered orally on daily basis for seven days followed by an additional dose 30 minutes before hepatic ischemia to study their hepatoprotective effects against hepatic ischemia (30 minutes)/reperfusion (30 minutes) injury. The degree of hepatic injury was assessed by measuring serum alanine transaminase (ALT) and aspartate transaminase (AST) activities, serum total, direct and indirect bilirubin (tBil, dBil and iBil) levels, liver weight/body weight ratio, hepatic myeloperoxidase (MPO) activity, hepatic contents of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) as well as histopathological changes. In the in vitro experiment, freshly isolated rat hepatocyte suspensions were prepared according to the collagenase perfusion method and divided into 16 flasks. Doses of test agents were selected according to the published literature as well as pilot trials. All flasks, except one left as normal control, were incubated with 5 mM CCl4. All other flasks, except the one left as CCl4 control, were incubated with test agents and/or with nitric oxide synthase (NOS) inhibitors N?-nitro-L-arginine methyl ester (L-NAME; a non-specific inhibitor) and aminoguanidine (AG; a specific inhibitor to inducible nitric oxide synthase or iNOS enzyme) 30 minutes before CCl4. Concentrations of the test agents used were as follows: NAC (5 mM), aloe extract (100 µl/ml), grape seed extract (100 µg/ml), nutmeg extract (100 µg/ml), L-NAME (5 mM) and AG (1 mM). Combinations between test agents and NOS inhibitors were also done as follows: NAC + L-NAME, NAC + AG, aloe + L-NAME, aloe + AG, grape seed + L-NAME, grape seed + AG, nutmeg + L-NAME and nutmeg + AG. Assessment of hepatocyte injury was performed by measuring percent lactate dehydrogenase (LDH) leakage, TBARS production, cellular GSH content, total nitrate/nitrite (NOX) production, cellular calcium ([Ca2+]i) content and cellular adenosine triphosphate (ATP) content. These parameters were measured in hepatocyte samples removed from the flasks at times -30, 0, 30, 60, 90 and 120 minutes of hepatocyte incubation with CCl4. The main findings of the present investigation can be summarized as follows: I. In vivo Experiments: 1. Daily administration of oral NAC (150, 300 or 600 mg/kg/day), aloe extract (5, 10, 20 ml/kg/day), grape seed extract (200, 400 or 800 mg/kg/day) or nutmeg extract (250, 500 or 1000 mg/kg/day) did not significantly affect serum ALT or AST activities of normal animals. 2. Hepatic ischemia for 15 minutes followed by reperfusion for 15, 30 or 60 minutes did not result in significant elevation of serum transaminases ALT and AST activities compared to sham-operated animals. 3. Hepatic ischemia for 30 minutes followed by 30 minutes of reperfusion significantly elevated serum transaminases ALT and AST compared to sham-operated animals and the elevation was even significantly higher when the reperfusion period was increased to 60 minutes. 4. Hepatic ischemia for 30 minutes followed by 30 minutes of reperfusion significantly increased serum tBil and dBil levels, increased hepatic content of TBARS, decreased hepatic stores of GSH, increased hepatic MPO activity and resulted in significant histopathological changes like inflammatory infiltration, blood congestion and loss of normal hepatic architecture. On the other hand, no significant effect was noted regarding iBil or liver weight/body weight ratio. 5. Pre-treatment of rats with NAC (300 mg/kg/day; p.o.), aloe extract (10 ml/kg/day, p.o.), grape seed extract (400 mg/kg/day, p.o.), nutmeg extract (500 mg/kg/day, p.o.) or a combination of aloe, grape seed and nutmeg extracts for 7 days, plus an additional dose 30 minutes before ischemia, significantly reduced hepatic IR-induced injury as evidenced by suppression of serum transaminases ALT and AST as well as elevation of hepatic GSH content. The histopathological findings strongly inforced the obtained results of the biochemical evaluations. 6. Pre-treatment of rats with NAC (300 mg/kg/day; p.o.), aloe extract (10 ml/kg/day, p.o.), or a combination of aloe, grape seed and nutmeg extracts significantly reduced hepatic MPO activity compared to ischemic control. 7. Pre-treatment of rats with NAC (300 mg/kg/day; p.o.), aloe extract (10 ml/kg/day, p.o.), grape seed extract (400 mg/kg/day, p.o.) or a combination of aloe, grape seed and nutmeg extracts for 7 days, plus an additional dose 30 minutes before ischemia, significantly reduced hepatic TBARS content compared to ischemic control. 8. On the other hand, neither of the test agents or their combination resulted in significant effects regarding serum tBil, dBil and iBil values or liver weight/body weight ratio. II. In vitro Experiment: 1. Incubation of freshly-isolated suspended rat hepatocytes with CCl4 significantly increased LDH leakage, TBARS production and [Ca2+]i content coupled with decreased cellular GSH content. These effects were observed as early as 30 minutes following CCl4 addition and continued till the end of the experiment. In addition, CCl4-incubation also significantly elevated NOX production and decreased cellular ATP content after 60, 90 and 120 minutes of incubation as compared to normal flasks. 2. Pre-incubation of CCl4-intoxicated hepatocytes with NAC significantly prevented CCl4-induced LDH leakage and significantly increased cellular GSH content after 30 minutes of addition of CCl4 and continued till the end of the experiment. It also restored TBARS production as well as [Ca2+]i and ATP contents after 60 minutes of addition of CCl4 and continued till the end of the experiment. NAC also significantly suppressed NOX production after 90 and 120 minutes of incubation with the hepatotoxin. 3. Pre-incubation of CCl4-intoxicated hepatocytes with aloe extract significantly suppressed CCl4-induced LDH leakage and [Ca2+]i elevation and significantly increased cellular GSH content after 60 minutes of addition of CCl4 and continued till the end of the experiment. It also suppressed TBARS and NOX production and increased ATP contents after 120 minutes of addition of CCl4. 4. Pre-incubation of CCl4-intoxicated hepatocytes with grape seed extract significantly prevented CCl4-induced LDH leakage and significantly increased cellular GSH content after 30 minutes of addition of CCl4 and continued till the end of the experiment. The extract significantly decreased [Ca2+]i content after 60 minutes of incubation with the hepatotoxin and continued till the end of the experiment. It also suppressed TBARS and NOX production and increased ATP contents after 90 and 120 minutes of addition of CCl4. 5. Pre-incubation of CCl4-intoxicated hepatocytes with nutmeg extract significantly suppressed CCl4-induced elevation of [Ca2+]i content after 30 minutes of addition of CCl4 till the end of the experiment. It also suppressed LDH leakage after 60 minutes of addition of CCl4 and continued till the end of the experiment. The extract significantly suppressed TBARS and NOX production and increased GSH and ATP contents after 90 and 120 minutes of incubation with the CCl4. 6. Pre-incubation of CCl4-intoxicated hepatocytes with L-NAME significantly increased LDH leakage, TBARS production and [Ca2+]i content and decreased GSH content after 30 minutes of incubation with CCl4. It significantly suppressed NOX after 30 minutes of incubation with CCl4 and the effect continued till the end of the experiment. 7. Pre-incubation of CCl4-intoxicated hepatocytes with AG significantly reduced LDH leakage, TBARS production and [Ca2+]i content and increased GSH and ATP contents after 120 minutes of incubation with the toxicant. It significantly suppressed NOX production after 90 and 120 minutes of incubation with CCl4. 8. Pre-incubation of CCl4-intoxicated hepatocytes with L-NAME plus NAC, aloe extract, grape seed extract or nutmeg extract significantly elevated LDH after 30 minutes (regarding aloe and nutmeg), 60 minutes (regarding NAC and grape seed) and 90 minutes (regarding NAC, aloe and grape seed), elevated TBARS after 90 minutes (regarding aloe and grape seed) and 120 minutes (regarding NAC and grape seed), suppressed NOX after 30, 60, 90 and 120 minutes (with all agents) and elevated [Ca2+]i after 60 minutes (regarding NAC and aloe) and 90 minutes (regarding grape seed) of incubation with CCl4 as compared to the respective controls not receiving L-NAME. 9. Pre-incubation of CCl4-intoxicated hepatocytes with AG plus aloe, grape seed or nutmeg (but not NAC) significantly suppressed NOX after 120 minutes of incubation with CCl4 as compared to the respective controls not receiving AG. The following conclusions could be deduced from the present investigation: 1. Hepatic IR injury and freshly isolated hepatocytes intoxiacated with CCl4 present good hepatotoxicity models in vivo and in vitro, respectively, for studying effects of different hepatoprotective agents. Free radical production, GSH and ATP depletion, inflammatory reactions, NO production and calcium deregulation are mainly involved. 2. N-acetylcysteine as well as aloe, grape seed and nutmeg extracts could be promising hepatoprotective agents by virtue of their anti-oxidant, anti-inflammatory, immunomodulatory, calcium channel blocking, liver microsomal enzyme inhibitory, cytosolic anti-oxidant enzyme stimulatory, anti-platelet, metal chelating and/or hemodynamic potentials. 3. Non-selective NOS inhibition by L-NAME potentiates CCl4-induced hepatotoxicity on freshly isolated rat hepatocytes and decreases the hepatoprotective effects of NAC as well as aloe, grape seed and nutmeg extracts. Alternatively, selective iNOS inhibition by AG reduces this toxicity without affecting the protective effects of the test agents. Possible expectation is that induction of iNOS is responsible for toxic production of massive amounts of nitric oxide (NO) through oxidative and nitrosative stress. On the other hand, constitutive nitric oxide synthase (cNOS) is responsible for the production of cytoprotective amounts of NO leading to favorable liver microsomal enzyme suppression and anti-oxidant potentials. According to the present study, natural extracts like aloe, grape seed and nutmeg could be promising hepatoprotective agents against different models of liver injury, and NO production might play a very important role in modulationg their protective effects.